Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O2 to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane1; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron2. Upon re-evaluation of these data, however, this proposal has been questioned3,4, and a transport mechanism including proton pumping in the reductive phase—that is, during the transfer of the first two electrons—was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase5. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 09 August 2018
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Wikström, M. K. Proton pump coupled to cytochrome c oxidase in mitochondria. Nature 266, 271–273 (1977)
Wikström, M. Identification of the electron transfers in cytochrome oxidase that are coupled to proton-pumping. Nature 338, 776–778 (1989)
Michel, H. The mechanism of proton pumping by cytochrome c oxidase. Proc. Natl Acad. Sci. USA 95, 12819–12824 (1998)
Michel, H. Cytochrome c oxidase: catalytic cycle and mechanisms of proton pumping—a discussion. Biochemistry 38, 15129–15140 (1999)
Verkhovsky, M. I., Jasaitis, A., Verkhovskaya, M. L., Morgan, J. E. & Wikström, M. Proton translocation by cytochrome c oxidase. Nature 400, 480–483 (1999)
Ferguson-Miller, S. & Babcock, G. T. Heme/copper terminal oxidases. Chem. Rev. 96, 2889–2907 (1996)
Michel, H., Behr, J., Harrenga, A. & Kannt, A. Cytochrome c oxidase: structure and spectroscopy. Annu. Rev. Biophys. Biomol. Struct. 27, 329–356 (1998)
Nilsson, T. Photoinduced electron transfer from tris(2,2′-bipyridyl)ruthenium to cytochrome c oxidase. Proc. Natl Acad. Sci. USA 89, 6497–6501 (1992)
Ruitenberg, M. et al. Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase. Proc. Natl Acad. Sci. USA 97, 4632–4636 (2000)
Mitchell, R. & Rich, P. R. Proton uptake by cytochrome c oxidase on reduction and on ligand binding. Biochim. Biophys. Acta 1186, 19–26 (1994)
Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376, 660–669 (1995)
Thomas, J. W., Puustinen, A., Alben, J. O., Gennis, R. B. & Wikström, M. Substitution of asparagines for asparate-135 in subunit I of the cytochrome bo ubiquinol oxidase of Escherichia coli eliminates proton-pumping activity. Biochemistry 32, 10923–10928 (1993)
Thomas, J. W., Lemieux, L. J., Alben, J. O. & Gennis, R. B. Site-directed mutagenesis of highly conserved residues in helix VIII of subunit I of the cytochrome bo ubiquinol oxidase from Escherichia coli: an amphipathic transmembrane helix that may be important in conveying protons to the binuclear center. Biochemistry 32, 11173–11180 (1993)
Verkhovsky, M. I., Tuukkanen, A., Backgren, C., Puustinen, A. & Wikström, M. Charge translocation coupled to electron injection into oxidized cytocrome c oxidase from Paracoccus denitrificans. Biochemistry 40, 7077–7083 (2001)
Witt, S. N. & Chan, S. I. Evidence for a ferryl Fea3 in oxygenated cytochrome c oxidase. J. Biol. Chem. 262, 1446–1448 (1987)
Vygodina, T. V. & Konstantinov, A. A. H2O2-Induced conversion of cytochrome c oxidase peroxy complex to oxoferryl state. Ann. NY Acad. Sci. 550, 124–138 (1988)
Moody, A. J. Ligation and electronation states of cytochrome c oxidase in relation to other oxidases and peroxidases. Biochem. Soc. Trans. 19, 617–622 (1991)
Greenwood, C., Wilson, M. T. & Brunori, M. Studies on partially reduced mammalian cytochrome oxidase. Reactions with carbon monoxide and oxygen. Biochem. J. 137, 205–215 (1974)
Brzezinski, P. & Malmström, B. G. The reduction of cytochrome c oxidase by carbon monoxide. FEBS Lett. 187, 111–114 (1985)
Oliveberg, M. & Malmström, B. G. Internal electron transfer in cytochrome c oxidase: evidence for a rapid equilibrium between cytochrome a and the bimetallic site. Biochemistry 30, 7053–7057 (1991)
Konstantinov, A. A., Siletsky, S., Mitchell, D., Kaulen, A. & Gennis, R. The roles of two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer. Proc. Natl Acad. Sci.USA 94, 9085–9090 (1997)
Jasaitis, A., Verkhovsky, M. I., Morgan, J. E., Verkhovskaya, M. L. & Wikström, M. Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen. Biochemistry 38, 2697–2706 (1999)
We thank the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and the Max-Planck-Gesellschaft for financial support. We are grateful to B. Ludwig for providing the D124N mutant.
About this article
Cite this article
Ruitenberg, M., Kannt, A., Bamberg, E. et al. Reduction of cytochrome c oxidase by a second electron leads to proton translocation. Nature 417, 99–102 (2002). https://doi.org/10.1038/417099a
This article is cited by
Nature Communications (2018)
Amino Acids (2013)
Archives of Microbiology (2008)
Role of cooperative H+/e− Linkage (redox Bohr effect) at heme a/CuA and heme a 3/CuB in the proton pump of cytochrome c oxidase
Biochemistry (Moscow) (2005)